Abstract
The generation of bidimensional neuronal cultures and tridimensional cerebral organoids is gaining exponential interest to investigate the causes of neurodegenerative diseases, as these models permit to study human cells and tissue, and to reconstruct in vitro the pathophysiology of the human brain.
In Parkinson’s disease (PD), dopamine neurons of the substantia nigra in the midbrain are lost and constitute the hallmark pathology. In addition, alpha-synuclein (aSyn) accumulation in Lewy pathology is found in several brain regions. For these reasons, human modelling focuses on the use of patient iPSCs to derive midbrain and cortical neurons, and generate midbrain and forebrain organoids.
Our group has recently investigated the small GTPase Rit2 as a novel target for disease modification in PD. We found Rit2 to interact with the PD-protein LRRK2 and that its forced expression reduces aSyn accumulation and limits dopamine neurodegeneration. Further, we observed that Rit2 modulates lysosome function as part of its physiological role.
On these bases, we decided to investigate the consequences of Rit2 gene deletion in human models, to study its physiological functions and determine if it is sufficient to induce PD-related phenotypes.
We engineered iPSCs via CRISPR-Cas9 to generate Rit2 KO cells and used them to derive neuron and organoid models. Midbrain organoids were generated and cultivated up to 55 days, and we noted a slower growth rate, in terms of size, for Rit2 KO organoids. Biochemically, we assessed the presence of TH, TPPP/p25 (for oligodendrocytes) and pS129-aSyn (which was trending to an increase). Confocal imaging revealed that dopamine neurons within organoids displayed a fragmented arborization, suggesting neuronal suffering, and distinct spots of pS129-aSyn signal (as opposed to diffuse staining in controls). Altogether, these data suggest that Rit2 KO sensitizes the midbrain to PD-like pathology. In addition, we plated the midbrain organoids for recording with Multiple Electrode Array (MEA), and observed that Rit2 KO causes a decrease in the mean firing rate of the neuronal population. Consistently, we differentiated Rit2 KO iPSCs into 2D dopamine neurons and performed whole-cell patch clamp electrophysiology. Rit2 KO neurons display a dramatic reduction of spontaneous action potential firing, indicating an impairment in neuronal transmission caused by Rit2 gene deletion. Interestingly, 2D dopamine neurons displayed a trend to reduced TH immunosignal, at the confocal analysis.
Our data indicate that Rit2 affects the maintenance and function of dopamine neurons in 2D and 3D human models, enhancing its relevance for PD. Currently, we are investigating cortical neurons and forebrain organoids to compare regional specificity of effects, and will be linking these phenotypes with lysosome function, as Rit2 is a regulator of this organelle.